Fibroblast growth factor 21 (
FGF21) is a promising
drug candidate for the treatment of
type 2 diabetes. However, the use of wild type native
FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based
protein engineering approach to generate a potent long-acting
FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion
protein was constructed by fusing the Fc domain of human
IgG1 to the N-terminus of human mature
FGF21 via a linker
peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native
FGF21. Two specific point mutations were introduced into
FGF21. The
leucine to
arginine substitution at position 98 (L98R) suppressed
FGF21 aggregation at high concentrations and elevated temperatures. The
proline to
glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of
FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type
protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native
FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in
blood glucose levels and
body weight gains up to 5-7 days, whereas the efficacy of
FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting
FGF21 analog with a favorable
pharmaceutical property for potential clinical development.